Detection of Circulating Tumor Cells Using Nanoparticle
4. Experimental section Chemicals and equipment
Rabbit polyclonal antibodies to EpCAM were purchased from Abnova (D01P) and from Abcam (ab65052), mouse monoclonal mouse antibody (B302(323/A3)) to EpCAM was purchased from Abcam (ab8601), and FITC-conjugated anti-rabbit antibody was purchased from Sigma (F0382). Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4.3H2O, 99.9%) and trisodium citrate (Na3C6H5O7.2H2O) were purchased from Sigma-Aldrich (Spain). Unless otherwise stated, all buffer reagents and other inorganic chemicals were supplied by Sigma-Aldrich (Spain). All chemicals were used as received
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and all aqueous solutions were prepared in double-distilled water. The phosphate buffer solution (PBS) was composed of 0.01M phosphate buffered saline, 0.137M NaCl, 0.003M KCl (pH 7.4). Samples for SEM analysis were prepared by using glutaraldehyde and hexamethyldisilazane (HMDS) microscopy grade solutions, Sigma-Aldrich (Spain).
A semi-automatic screen-printing machine DEK248 (DEK International, Switzerland) was used for the fabrication of the screen printed carbon electrodes (SPCEs). The electrodes were printed over Autostat HT5 polyester sheet (McDermid Autotype, UK) using Electrodag 423SS carbon ink for working and counter electrodes, Electrodag 6037SS silver/silver chloride ink for reference electrode and Minico 7000 Blue insulating ink (Acheson Industries, The Netherlands) to insulate the contacts and define the sample interaction area.
The electrochemical experiments where performed with a µAutolab II (Echo Chemie, The Netherlands) potentiostat/galvanostat connected to a PC and controlled by Autolab GPES software. All measurements were carried out at room temperature, with a working volume of 50µL, which was enough to cover the three electrodes contained in the home made SPCE used as electrotransducer, connected to the potentiostat by a home made edge connector module.
Flow cytometry analysis of cells was undertaken with a BD FACSCalibur, Becton Dickinson. For optical microscopy analysis an Olympus IX85 motorized inverted microscope was used and SEM analysis was undertaken with a Merlin®FE-SEM.
Cell culture
Since the CTCs detach from a primary tumor we chose an adherent tumoral cell line, Human
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M. Maltez-da Costa et al. Detection of Circulating Tumor Cells Using Nanoparticles
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Colon Adenocarcinoma Cell line (Caco2), as a model for CTCs. Caco2 cells (European Collection of Cells Culture, No: 86010202) were maintained in Earle’s MEM supplemented with 10%(v/v) foetal bovine serum (FBS) and 2 mM L-glutamine. Cells were grown in a humidified incubator (95% air and 5% CO2 at 37ºC). Adherent cells in exponential phase were harvested by treatment with trypsin in order to detach the cells from the growth surface.
Human monocytes (THP-1), which grow in suspension, were used as blank and obtained from ECACC (88081201) and cultivated at 37ºC in a 5% CO2atmosphere.
Synthesis and biofunctionalization of gold nanoparticles
The 20-nm AuNPs were synthesized by an adapted method of the one pioneered by Turkevich et al. A total of 50 mL of 0.01% HAuCl4 solution was heated with vigorous stirring and 1.25mL of a 1% trisodium citrate solution was added quickly to the boiling solution. When the solution turned deep red, indicating the formation of gold nanoparticles, it was left stirring and cooling down. In this way, a dispersed solution of near 20-nm AuNPs was obtained. . The conjugation of AuNPs to anti-EpCAM antibody was performed according to the following procedure, previously optimized by our group. AuNPs suspension (1mL) was mixed with 100 µL of 100 µg/mL antibody solution and incubated at 25ºC for 20 min with gentle stirring. Subsequently, a blocking step with 5% BSA for 20 min at 25ºC was undertaken. Finally, a centrifugation at 14000 rpm and 5ºC, for 20 min was carried out and the AuNPs/anti-EpCAM conjugate was reconstituted in PBS-BSA (0.1%) solution and kept at 4ºC.
Microscopy images and cytometry analysis of cell interaction with biofunctionalized AuNPs
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A fluorescent tagged secondary antibody that recognizes anti-EpCAM antibody allowed the detection of AuNPs/anti-EpCAM-conjugate at the cell membrane by both fluorescence microscopy imaging and flow cytometry analysis. The preparation of samples for both methods was the same. Prior to the incubation, cells (suspension with 1x 106 cells.mL-1) were isolated from the culture medium by centrifugation (1000 rpm, 5min) and the pellet was resuspended in PBS-BSA 0.1%. In both methods, two samples of 2x 105 cells were incubated with 50µL of AuNPs/anti-EpCAM-conjugate, as prepared solution. After incubation (30 minutes/ 25ºC, with agitation) labeled cells were centrifuged, washed two times to eliminate the excess of anti-EpCAM functionalized AuNPs and redispersed in buffer. After washing by centrifugation the pellet was resuspended and incubated with FITC-conjugated anti-rabbit secondary antibody used as a label for fluorescence analysis. Controls were performed with citrate modified AuNPs without anti-EpCAM antibody, and also with AuNPs conjugated to another antibody that proved to be non-specific to Caco2 cells.
Electrochemical detection of AuNPs labeled Caco2 cells and selectivity test
The electrochemical detection of Caco2 cells based on the electrocatalytic detection of AuNP labeled anti-EpCAM was performed in HCl 1M by chronoamperometry. Samples were prepared by incubation of different amounts of Caco2 cells (from 0 to 1.5x 105 Caco2 cells) with 50µL of AuNPs/anti-EpCAM conjugate (30 minutes, 25ºC, with agitation). After removing the excess of AuNPs by centrifugation washing steps, samples were analyzed by chronoamperometry.
The samples used in the selectivity test were prepared by mixing in suspension the Caco2 cells with monocytes (THP-1 cells) in different proportions. They were then incubated with
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M. Maltez-da Costa et al. Detection of Circulating Tumor Cells Using Nanoparticles
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the AuNPs/anti-EpCAM conjugate (50µL of AuNPs/anti-EpCAM conjugate, 30 minutes, 25ºC, with agitation). After removing the excess of AuNPs by centrifugation washing steps, samples were detected by the electrochemical method described above. Samples with 100, 70, 50, 20 and 0 % of Caco2 cells were tested with the 100% corresponding to 5x 104 cells (0% of Caco2 means that the sample had only monocytes).
Scanning Electron Microscopy (SEM) images of cel interaction with biofunctionalized AuNPs
The technical advances in electron microscopy allow the achievement of excelent characterization tools both in the micro and nano domain. Scanning Electron Microscopy (SEM) is a well known characterization technique for cells, which have relative large dimensions, but the interactions of cells with small nanometer sized materials in liquid suspension its not so easy to observe. Cells often lack the requirements of structure stability and electron conductivity necessary for high magnification SEM images, and its often necessary to apply metalization procedures that cover all the sample with a nano/micro layer of material that would hide the low nanometer rugosity of small nanoparticles interacting with the cell surface, in addition to changing its outer-layer chemical composition.
The accurate characterization of the interaction Caco2 cell-biofunctionalized AuNPs is very important to elucidate the specifity and selectivity of the sensing system presented here.
Therefore, after incubation of cell samples with anti-EpCAM functionalized-AuNPs as described above, the cells were kept in suspension and treated with glutaraldehyde solution folowed by sequential ethanol solutions with increasing purity, and they were finally resuspended in HMDS solution. This protocol allows a good fixation of cells in suspension while mantaining cell shape and the membrane outer structure, and proved to be well suited
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for the observation of cells without the need of metalization or any other procedure that would change or mask the nanosized conjugate (AuNPs/anti-EpCAM) used to label the cell membrane.
Acknowledgements
We acknowledge MICINN (Madrid) for the projects PIB2010JP-00278 and IT2009-0092, the E.U.’s support under FP7 contract number 246513 ‘‘NADINE’’ and the NATO Science for Peace and Security Programme’s support under the project SfP 983807.
We also thank the SCAC-IBB members: Manuela Costa, for the technical support on cytometry experiments and data analysis, Francisca Garcia and Francisco Cortes, for the technical support on cell culture; The Servei de Microscòpia UAB members: Onofre Castell and Marcos Rosado for the technical support with SEM imaging and for the important inputs on the sample preparation protocols.
[1] T. G. Lugo, S. Braun, R. J. Cote, K. Pantel, V. Rusch, Journal of clinical oncology!:
official journal of the American Society of Clinical Oncology 2003, 21, 2609-15.
[2] S. Nagrath, L. V. Sequist, S. Maheswaran, D. W. Bell, D. Irimia, L. Ulkus, M. R.
Smith, E. L. Kwak, S. Digumarthy, A. Muzikansky, P. Ryan, U. J. Balis, R. G.
Tompkins, D. a Haber, M. Toner, Nature 2007, 450, 1235-9.
[3] P. Paterlini-Brechot, N. L. Benali, Cancer letters 2007, 253, 180-204.
[4] K. Pantel, C. Alix-Panabières, Trends in molecular medicine 2010, 16, 398-406.
[5] N. Bednarz-Knoll, C. Alix-Panabières, K. Pantel, Breast cancer research!: BCR 2011, 13, 228.
[6] B. Taback, a D. Chan, C. T. Kuo, P. J. Bostick, H. J. Wang, a E. Giuliano, D. S. Hoon, Cancer research 2001, 61, 8845-50.
Submitted to
M. Maltez-da Costa et al. Detection of Circulating Tumor Cells Using Nanoparticles
- 15 -
[7] S. Wang, H. Wang, J. Jiao, K.-J. Chen, G. E. Owens, K.-ichiro Kamei, J. Sun, D. J.
Sherman, C. P. Behrenbruch, H. Wu, H.-R. Tseng, Angewandte Chemie (International ed. in English) 2009, 48, 8970-3.
[8] J. den Toonder, Lab on a chip 2011, 11, 375-7.
[9] K. Li, R. Zhan, S.-S. Feng, B. Liu, Analytical Chemistry 2011, 83, 2125-2132.
[10] M. Perfézou, A. Turner, A. Merkoçi, Chemical Society reviews 2011, DOI 10.1039/c1cs15134g.
[11] P. T. H. Went, A. Lugli, S. Meier, M. Bundi, M. Mirlacher, G. Sauter, S. Dirnhofer, Human Pathology 2004, 35, 122-128.
[12] C. Patriarca, R. M. Macchi, A. K. Marschner, H. Mellstedt, Cancer treatment reviews 2011, DOI 10.1016/j.ctrv.2011.04.002.
[13] L. Belov, J. Zhou, R. I. Christopherson, Cell Surface Markers in Colorectal Cancer Prognosis., 2010.
[14] J. Wang, Small (Weinheim an der Bergstrasse, Germany) 2005, 1, 1036-43.
[15] J. Das, M. A. Aziz, H. Yang, Journal of the American Chemical Society 2006, 128, 16022-3.
[16] D. Tang, R. Yuan, Y. Chai, Analytical chemistry 2008, 80, 1582-8.
[17] D. a Giljohann, D. S. Seferos, W. L. Daniel, M. D. Massich, P. C. Patel, C. a Mirkin, Angewandte Chemie (International ed. in English) 2010, 49, 3280-94.
[18] M. Pumera, M. T. Castañeda, M. I. Pividori, R. Eritja, A. Merkoçi, S. Alegret, Langmuir!: the ACS journal of surfaces and colloids 2005, 21, 9625-9.
[19] A. Ambrosi, M. T. Castañeda, A. J. Killard, M. R. Smyth, S. Alegret, A. Merkoçi, Analytical chemistry2007, 79, 5232-40.
[20] M. Maltez-da Costa, A. De La Escosura-Muñiz, A. Merkoçi, Electrochemistry Communications 2010, 12, 1501-1504.
[21] A. De La Escosura-Muñiz, M. Maltez-da Costa, C. Sánchez-Espinel, B. Díaz-Freitas, J.
Fernández-Suarez, Á. González-Fernández, A. Merkoçi, Biosensors and Bioelectronics 2010, 26, 1710-1714.
[22] A. De La Escosura-Muñiz, C. Sánchez-Espinel, B. Díaz-Freitas, A. González-Fernández, M. Maltez-da Costa, A. Merkoçi, Analytical Chemistry 2009, 81, 10268-10274.
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[23] J. Turkevich, P. C. Stevenson, J. Hillier, 1953, 57, 670-673.
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Figure 1.(a) Scheme of the CaCo2 cells biorecognition with AuNPs/anti-EpCAM-antibodies
and further detection through the Hydrogen Evolution Reaction (HER) electrocatalyzed by the AuNPs labels; b) Left: Chronoamperograms registered in 1M HCl, during the HER applying a constant voltage of -1.0V, for AuNPs-labeled CaCo2 cells (3.5 x104 - red curve) and for the control (PBS/BSA - blue curve). Right: Comparison of the corresponding analytical signals (absolute value of the current registered at 50 seconds) of the blank and sample.
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Figure 2. TEM images of AuNPs, before (a) and after (b) biofunctionalization with anti--EpCAM antibody. (c) UV-Vis spectra of AuNPs before ( ) and after ( ) biofunctionalization with anti-EpCAM.
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Figure 3. (a, b) Microscopy imaging of Caco2 cells incubated sequentially with
AuNPs/anti-EpCAM and a FITC conjugated secondary anti-rabbit antibody, at bright field a) and fluorescence mode b). (c) Flow cytometry analysis of Caco2 cells labeled with AuNPs/anti-EpCAM. Histogram count of unlabeled (black) vs. labeled (red) cells using the same FITC secondary antibody as in 2a, b).
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Figure 4. Electrochemical results obtained for increasing number of Caco2 cells after
incubation with AuNPs/anti-EpCAM (a) and for mixed suspensions of Caco2 and monocytes (THP-1 cells) at different Caco2/THP-1 ratios (total cells amount: 5 x104) after incubation with AuNPs/anti-EpCAM (b).
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Figure 5. Right: SEM-Backscattered images of the cell surfaces of (a): Caco2 cells before
incubation with AuNPs/anti-EpCAM; (b) Caco2 and (c) monocytes after incubation with AuNPs/anti-EpCAM in the same sample. The zoom in (b) corresponds to a detail of AuNPs/anti-EpCAM at CaCo2 surface (insets of scattered images of the same cell area are also shown for comparison purposes). Left: SEM full images of Caco2 and monocytes and the corresponding schematic cartoons.
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A novel electrochemical strategy to detect and quantify Circulating Tumor Cells (CTCs) based on the selective labeling with electrocatalytic nanoparticles has been achieved. The proposed sensor is a rapid and simple detection device that uses specific antibody/AuNPs conjugate to recognize tumor cells in suspension followed by fast electrochemical detection in a user-friendly platform.
TOC Keyword:
M. Maltez-da Costa, A. de la Escosura-Muñiz, C. Nogués, L. Barrios, E. Ibañez, A. Merkoçi Detection of Circulating Tumor Cells Using Nanoparticles
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Supporting information
Detection of Circulating Tumor Cells Using Nanoparticles **
Marisa Maltez-da Costa, Alfredo de la Escosura-Muñiz, Carme Nogués, Leonard. Barios, Elena Ibáñez, Arben Merkoçi *
[*] Prof. Arben Merkoçi
ICREA, Institució Catalana de Recerca i Estudis Avançats and Nanobioelectronics &
Biosensors Group, CIN2 (ICN-CSIC), Catalan Institute of Nanotechnology, Campus de la UAB Bellaterra (Barcelona), 08193 Spain
E-mail: arben.merkoci.icn@uab.es
Marisa Maltez-da Costa, Dr. Alfredo de la Escosura-Muñiz
Nanobioelectronics & Biosensors Group, CIN2 (ICN-CSIC), Catalan Institute of Nanotechnology, Campus de la UAB Bellaterra (Barcelona), 08193 Spain Prof. Carmen Nogués, Prof. Leonard Barrios, Dr. Elena Ibáñez
Departament de Biologia Cel•lular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, Campus UAB-Facultat de Biociències
Scanning Electron Microscopy (SEM) images of cell interaction with biofunctionalized AuNPs
In Figure S1, we can see several SEM images of Caco2 cells. In the images acquired with higher magnification (a-d) it is possible to observe the cell membrane both before (a,c) and after (b,d) Caco2 incubation with anti-EpCAM-functionalized AuNPs. In image b we can observe the cell membrane with enough detail to discriminate the small nanoparticles attached. To be sure that this small structures are anti-EpCAM-functionalized AuNPs we used the Backscattered Electrons mode (BSE) to diferentiate between elements (c,d). Since heavy elements backscatter electrons more strongly than light elements, they appear brighter in the image enhancing the contrast between different chemical compositions. In the image from
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Caco2 sample incubated with AuNPs (d) the nanoparticles are visualized wit a much better contrast indicating the presence of a much heavier nanosized element than the background.
The samples used in the selectivity test were also characterized by SEM using the same sample preparation protocol. In Figure S2 we can see SEM images obtained for the sample containing 70% of Caco2 and 30% of monocytes. Both cells have a round shape which makes it difficult to differentiate them by optical microscopy techniques. But with the optimized SEM preparation protocol we obtained high quality images where we can observe the detail of the plasma membrane and, using the Backscattered Electrons mode (BSE), observe the presence of anti-EpCAM-functionalized AuNPs only at the surface of Caco2 cells. No AuNPs were found in the several monocytes present in the sample and all the Caco2 cells displayed numerous particles all around the cell surface.
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Figure S1: SEM images of Caco2 cells. Full cell image (e) and higher magnification images of cell membrane before (a,c) and after (b,d) their incubation with anti-EpCAM-functionalized AuNPs . Images c and d were acquired with backscatered electrons mode.
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Figure S2: SEM images for the samples containing monocytes (THP-1) and Caco2 cells.
Images showing a THP-1 cell (e) and a Caco2 cell (f) and higher magnification images of cell plasma membrane from THP-1 (a,c) and Caco2 (b,d) after incubation with anti-EpCAM-functionalized AuNPs. Images c and d were acquired with backscattered electrons mode.